PerCP-Cy™5.5 Mouse anti-Stat1 (pY701)

BD Phosflow™ PerCP-Cy™5.5 Mouse anti-Stat1 (pY701)

Name: PerCP-Cy™5.5 Mouse anti-Stat1 (pY701)
Brand: BD Phosflow™
SKU: 560113

Clone 4a

(RUO)

Analysis of Stat1 (pY701) in human peripheral blood monocytes. Peripheral blood mononuclear cells (PBMC) were either left unstimulated (unshaded) or stimulated (shaded) with 100 ng/ml (final concentration) BD Pharmingen™ Recombinant Human IFNγ (Cat. No. 554617) for 15 minutes at 37°C. The PBMC were fixed (BD Cytofix™ Fixation buffer, Cat. No. 554655) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes and then stained with PerCP-CY™5.5 anti-Stat1 (pY701). For data analysis, monocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSCanto™ II flow cytometer.

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Product Details

Brand: BD Phosflow™

Reactivity: Human (QC Testing), Mouse (Reactivity Confirmed in Development), Rat (Predicted)

Isotype: Mouse IgG2a

Immunogen: Phosphorylated Human Stat1 Peptide

Application: Intracellular staining (flow cytometry) (Routinely Tested)

Vol. Per Test: 20 µl

RRID: AB_1645550

Storage Buffer: Aqueous buffered solution containing BSA and ≤0.09% sodium azide.

Regulatory Status: RUO

Preparation And Storage

The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with PerCP-Cy5.5 under optimum conditions, and unconjugated antibody and free PerCP-Cy5.5 were removed. Storage of PerCP-Cy5.5 conjugates in unoptimized diluent is not recommended and may result in loss of signal intensity. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Recommended Assay Procedures

Either BD Cytofix™ fixation buffer or BD Phosflow™ Fix Buffer I may be used for cell fixation.

This mAb was characterized by flow cytometry (Flow) and western blot analysis (WB) using these model systems:

Method Species Cells Treatment Fixation Perm buffer Result

Flow Human PBMC IFNγ Fixation Buffer III Positive Staining

Flow Human PBMC IFNγ Fixation Buffer I or II Unsatisfactory

WB Human U937 Cell Lysate IFNγ Not Applicable Not Applicable 91/84 kDa

WB Human A431 Cell Lysate EGF Not Applicable Not Applicable 91/84 kDa

Product Notices

This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
PerCP-Cy5.5–labelled antibodies can be used with FITC- and R-PE–labelled reagents in single-laser flow cytometers with no significant spectral overlap of PerCP-Cy5.5, FITC, and R-PE fluorescence.
PerCP-Cy5.5 is optimized for use with a single argon ion laser emitting 488-nm light. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using dual-laser cytometers, which may directly excite both PerCP and Cy5.5™. We recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
This product is subject to proprietary rights of Amersham Biosciences Corp. and Carnegie Mellon University and made and sold under license from Amersham Biosciences Corp. This product is licensed for sale only for research. It is not licensed for any other use. If you require a commercial license to use this product and do not have one return this material, unopened to BD Biosciences, 10975 Torreyana Rd, San Diego, CA 92121 and any money paid for the material will be refunded.
Cy is a trademark of Amersham Biosciences Limited. This conjugated product is sold under license to the following patents: US Patent Nos. 5,486,616; 5,569,587; 5,569,766; 5,627,027.
Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
Source of all serum proteins is from USDA inspected abattoirs located in the United States.
For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.

Companion Products

Fixation Buffer RUO

Size 100 mL Cat No. 554655

Fix Buffer I RUO

Size 250 mL Cat No. 557870

Perm Buffer III RUO

Size 125 mL Cat No. 558050

Recombinant Human IFN-γ RUO

Size 50 µg Cat No. 554617

560113 Rev. 3

Antibody Details

Stat (Signal transducer and activators of transcription) proteins mediate the biological activity of cytokines, including interleukins, interferons, erythropoietin, and growth factors. Ligand-receptor interaction activates constitutively-associated JAK family kinases as well as subsequent recruitment and activation of Stat proteins by tyrosine phosphorylation. Active Stat proteins then move to the nucleus to promote transcription of cytokine-inducible genes. Seven Stat proteins have been cloned, each of which is differentially expressed and/or activated in a cytokine-specific and cell type-specific manner. Stat1 and Stat2 are components of the ISGF3 (Interferon-Stimulated Gene Factor 3) complex, the primary transcription activator induced by interferon binding to a specific cell-surface receptor. Stat1 has two alternatively spliced isoforms, 91-kDa Stat1α and 84-kDa Stat1β; Stat1α has 38 additional C-terminal amino acids. In response to the binding of IFNα, IFNγ, EGF, PDGF, or CSF-1 to their respective receptors, the Stat1 subunits become tyrosine-phosphorylated at Y701, and the complex translocates to the nucleus. This forms an active complex that includes the DNA-binding p48 subunit, and is responsible for modulating interferon-stimulated genes (ISGs) transciption.

The 4a monoclonal antibody recognizes the phosphorylated Y701 in Stat1α and Stat1β.

560113 Rev. 3

Format Details

PerCP-Cy5.5

PerCP-Cy5.5 dye is part of the BD blue family of dyes. This tandem fluorochrome is comprised of a fluorescent protein complex (PerCP) with an excitation maximum (Ex Max) of 482 nm and an acceptor dye with an emission maximum (Em Max) at 676 nm. PerCP-Cy5 is designed to be excited by the blue laser (488-nm) and detected using an optical filter centered near 680 nm (e.g., a 695/40 nm bandpass filter). The donor dye can be partially excited by the Violet (405-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.

Format: PerCP-Cy5.5

Excitation Source: Blue 488 nm

Excitation Max: 482 nm

Emission Max: 676 nm

560113 Rev.3

Citations & References

Development References (3)

Bromberg J, Darnell JE. The role of STATs in transcriptional control and their impact on cellular function. Oncogene. 2000; 19(21):2468-2473. (Biology). View Reference
Darnell JE Jr. STATs and gene regulation. Science. 1997; 277(5332):1630-1635. (Biology). View Reference
Fu XY, Zhang JJ. Transcription factor p91 interacts with the epidermal growth factor receptor and mediates activation of the c-fos gene promoter. Cell. 1993; 74(6):1135-1145. (Biology). View Reference

560113 Rev. 3

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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described

Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.